DISTINCT BEHAVIOR OF CARDIAC MYOSIN HEAVY-CHAIN GENE CONSTRUCTS INVIVO - DISCORDANCE WITH INVITRO RESULTS

Transcriptional thyroid hormone responsiveness of the cardiac alpha-my osin heavy chain (alpha-MHC) gene has been demonstrated in transfectio ns into fetal and neonatal cardiomyocytes and in transgenic mice. Howe ver, the correspondence between the regulation of MHC expression in di ssociated cells with that in the intact heart is unclear. Given the co st and time involved in generating multiple transgenic lines for the c haracterization of gene regulatory elements, we used direct cardiac ge ne transfer to identify elements regulating both basal and thyroid hor mone responsive cardiac alpha-MHC gene expression in the adult heart i n vivo. Sequences upstream of the rat alpha-MHC gene linked to a lucif erase reporter gene were injected into the hearts of adult rats subjec ted to various thyroid manipulations. The 161-bp sequence upstream of the transcription start site, which contains a TATA box, a CCAATT box, and a thyroid hormone response element, was transcriptionally active but not thyroid hormone responsive. The expression of a construct cont aining 388 bp of upstream sequence was increased by thyroid hormone ad ministration, a response that required an intact thyroid hormone respo nse element. However, expression of this construct failed to decrease to basal levels in a hypothyroid state. To confer complete (positive a nd negative) thyroid hormone regulation, 2,936 bp of upstream sequence was sufficient. These results demonstrate that, although necessary, t he thyroid hormone response element is not sufficient for complete thy roid hormone regulation of this gene in vivo. In addition, DNA sequenc es regulating the quantitative expression of cardiac alpha-MHC in the euthyroid state have been demonstrated. One sequence, an MEF-2 site, w hich has been shown to be essential for high levels of expression of a t least one other cardiac gene in neonatal cardiocytes, was mutated an d found not to affect alpha-MHC expression in the adult heart. These d ata emphasize the complexity of gene regulation in an intact organ, as pects of which cannot be simulated in culture.